During the previous funding period, we made significant advances in the discovery and characterization of the lipid rafts of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Cholesterol glycolipids form lipid rafts in both in vitro and host-derived spirochetes and that these domains have all the hallmarks of eukaryotic lipid rafts. We further demonstrated that the lipid rafts of B. burgdorferi shift their morphology at different temperatures and have a selective presence of lipoproteins, proteases and their substrates, sensing molecules, and homologs of eukaryotic lipid raft markers. Lipid rafts are dynamic structures with shifting protein cargo that define their biological functions. Fo this renewal application, we will expand our investigations into the dynamic features of the lipid rafts and into their function. The overall hypothesis is that the protein content as well the proten concentrations of lipid rafts change in response to the different conditions that the Borrelia moves through in its vector (tick) - host (mammal) stages, and that these changes influence the adaptation mechanisms required by the organisms to complete their life-cycle. To test this hypothesis, this renewal application will address the following Specific Aims: 1. To characterize the proteomes of the lipid rafts of Borrelia under different biological conditions, and 2. To probe the mechanisms of dynamic change and life-cycle adaptation by studying the contributions of unique elements of the proteome. A number of novel biophysical, imaging and biochemical approaches will provide the results to demonstrate that the Borrelia lipid rafts have important biological functions. Cholesterol as a component of bacterial membranes is being reported in an enlarging number of bacterial pathogens thereby increasing the significance of this proposal, as it is clear that cholesterol utilization is not an isolated feature of the Borreliae.